Genomic insights into the evolution, pathogenicity, and extensively drug-resistance of emerging pathogens Kluyvera and Phytobacter.

Introduction: Kluyvera is a Gram-negative, flagellated, motile bacillus within the Enterobacteriaceae. The case reports of clinical infections shed light on the importance of this organism as an emerging opportunistic pathogen. The genus Phytobacter, which often be misidentified with Kluyvera, is also an important clinically relevant member of the Enterobacteriaceae. However, the identification of Kluyvera and Phytobacter is problematic, and their phylogenetic relationship remains unclear. Methods: Here, 81 strains of Kluyvera and 16 strains of Phytobacter were collected. A series of comparative genomics approaches were applied to the phylogenetic relationship reconstruction, virulence related genes profiles description, and antibiotic resistance genes prediction. Results: Using average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH), we offered reliable species designations of 97 strains, in which 40 (41.24%) strains were incorrectly labeled. A new Phytobacter genomospecies-1 were defined. Phytobacter and Kluyvera show great genome plasticity and inclusiveness, which may be related to their diverse ecological niches. An intergenomic distances threshold of 0.15875 was used for taxonomy reassignments at the phylogenomic-group level. Further principal coordinates analysis (PCoA) revealed 11 core genes of Kluyvera (pelX, mdtL, bglC, pcak-1, uhpB, ddpA-2, pdxY, oppD-1, cptA, yidZ, csbX) that could be served as potential identification targets. Meanwhile, the Phytobacter specific virulence genes clbS, csgA-C, fliS, hsiB1_vipA and hsiC1_vipB, were found to differentiate from Kluyvera. We concluded that the evolution rate of Kluyvera was 5.25E-6, approximately three times higher than that of Phytobacter. Additionally, the co-existence of ESBLs and carbapenem resistance genes were present in approximately 40% strains, suggesting the potential development of extensively drug-resistant or even fully drug-resistant strains. Discussion: This work provided a better understanding of the differences between closely related species Kluyvera and Phytobacter. Their genomes exhibited great genome plasticity and inclusiveness. They not only possess a potential pathogenicity threat, but also a risk of multi-drug resistance. The emerging pathogens Kluyvera and Phytobacter warrant close attention.

Mechanism for transmission and pathogenesis of carbapenem-resistant Enterobacterales harboring the carbapenemase IMP and clinical countermeasures.

Carbapenem-resistant Enterobacterales (CRE) are some of the most important pathogens causing infections, which can be challenging to treat. We identified four blaIMP-carrying CRE isolates and collected clinical data. The transferability and stability of the plasmid were verified by conjugation, successive passaging, and plasmid elimination assays. The IncC blaIMP-4-carrying pIMP4-ECL42 plasmid was successfully transferred into the recipient strain, and the high expression of traD may have facilitated the conjugation transfer of the plasmid. Interestingly, the plasmid showed strong stability in clinical isolates. Whole-genome sequencing was performed on all isolates. We assessed the sequence similarity of blaIMP -harboring plasmid from our institution and compared it to plasmids for which sequence data are publicly available. We found that four blaIMP-carrying CRE belonged to four different sequence types. The checkerboard technique and time-kill assays were used to investigate the best antimicrobial therapies for blaIMP-carrying CRE. The time-kill assay showed that the imipenem of 1× minimum inhibitory concentration (MIC) alone had the bactericidal or bacteriostatic effect against IMP-producing strains at 4-12 h in vitro. Moreover, the combination of tigecycline (0.5/1/2 × MIC) and imipenem (0.5/1 × MIC) showed a bactericidal effect against the blaIMP-26-carrying CRECL60 strain.IMPORTANCECarbapenem-resistant Enterobacterales (CRE) are an urgent public health threat, and infections caused by these microorganisms are often associated with high mortality and limited treatment options. This study aimed to determine the clinical features, molecular characteristics, and plasmid transmissible mechanisms of blaIMP carriage as well as to provide a potential treatment option. Here, we demonstrated that conjugated transfer of the IncC blaIMP-4-carrying plasmid promotes plasmid stability, so inhibition of conjugated transfer and enhanced plasmid loss may be potential ways to suppress the persistence of this plasmid. The imipenem alone or tigecycline-imipenem combination showed a good bactericidal effect against IMP-producing strains. In particular, our study revealed that imipenem alone or tigecycline-imipenem combination may be a potential therapeutic option for patients who are infected with IMP-producing strains. Our study supports further trials of appropriate antibiotics to determine optimal treatment and emphasizes the importance of continued monitoring of IMP-producing strains in the future.

Pathotype Characterization of Plasmodiophora brassicae by European Clubroot Differential and Williams Sets in China.

Clubroot disease caused by the soil-borne Plasmodiophora brassicae is devastating to Brassicaceae crops and spreading rapidly in China in recent years, resulting in great yield losses annually. Virulence of P. brassicae populations specializes and is in dynamic change in the fields. Information on the pathotypes and their distributions is crucial to control the clubroot disease. Presently, the pathotypes of P. brassicae prevalent in China, however, are not well determined. In this study, we used 16 Brassica hosts, including the European Clubroot Differential (ECD) and Williams sets, to designate the pathotypes of 33 P. brassicae populations from 13 provinces. The 33 P. brassicae populations could be divided into 26 pathotypes by the ECD set or seven pathotypes by the Williams set, revealing ECD16/15/31 and ECD16/31/31 or P4 and P2 as the predominant pathotypes. We found that the Brassica rapa differentials ECD01 to ECD04 showed stable and high levels of resistance to most pathotypes of P. brassicae in China, thereby providing valuable resources for clubroot-resistance breeding of Brassicaceae crops. The ECD set exhibited much higher discernibility and further divided the isolates that belonged to the P4 pathotype into 10 ECD pathotypes. Isolates of ECD16/23/31 and ECD16/15/31 were strongly virulent on Huashuang 5R, the first and widely used clubroot-resistant cultivar of oilseed rape in China. As we learn, 26 pathotypes are the most diverse populations of P. brassicae characterized until now in China. Our study provides new insights into virulence specialization of P. brassicae and their geographical distributions, contributing to exploitation of clubroot-resistant resources and the field layout of the present resistant Brassica crops in China.

Anti-freezing dual-network hydrogels with high-strength, self-adhesive and strain-sensitive for flexible sensors.

Hydrogels as flexible sensor have attracted significant attention due to its conductivity, stretchability and flexibility. However, it is still a great challenge to prepare hydrogels that simultaneously possess high strength, anti-fatigue, self-adhesion, and anti-freezing. Herein, a multifunctional dual-network hydrogel was prepared by in situ polymerization of acrylic monomer in chitosan chains, and coordinated with aluminum chloride and glycerol. Based on chain entanglement, hydrogen bonding and coordination interactions, this dual-network hydrogel exhibited excellent mechanical properties, good fatigue resistance, and excellent adhesion performance. It can be used as a strain sensor for its stable conductivity and high sensitivity, which could monitor both large human motions and subtle motions. Due to the presence of glycerol, the hydrogel showed outstanding freezing resistance and still kept flexible and conductive even at low temperatures (-20 °C). This hydrogel can be applied as a flexible wearable sensor for monitoring human motion in extreme low-temperature condition.

Alternative splicing reprogramming in fungal pathogen Sclerotinia sclerotiorum at different infection stages on Brassica napus.

Alternative splicing (AS) is an important post-transcriptional mechanism promoting the diversity of transcripts and proteins to regulate various life processes in eukaryotes. Sclerotinia stem rot is a major disease of Brassica napus caused by Sclerotinia sclerotiorum, which causes severe yield loss in B. napus production worldwide. Although many transcriptome studies have been carried out on the growth, development, and infection of S. sclerotiorum, the genome-wide AS events of S. sclerotiorum remain poorly understood, particularly at the infection stage. In this study, transcriptome sequencing was performed to systematically explore the genome-scale AS events of S. sclerotiorum at five important infection stages on a susceptible oilseed rape cultivar. A total of 130 genes were predicted to be involved in AS from the S. sclerotiorum genome, among which 98 genes were differentially expressed and may be responsible for AS reprogramming for its successful infection. In addition, 641 differential alternative splicing genes (DASGs) were identified during S. sclerotiorum infection, accounting for 5.76% of all annotated S. sclerotiorum genes, and 71 DASGs were commonly found at all the five infection stages. The most dominant AS type of S. sclerotiorum was found to be retained introns or alternative 3' splice sites. Furthermore, the resultant AS isoforms of 21 DASGs became pseudogenes, and 60 DASGs encoded different putative proteins with different domains. More importantly, 16 DASGs of S. sclerotiorum were found to have signal peptides and possibly encode putative effectors to facilitate the infection of S. sclerotiorum. Finally, about 69.27% of DASGs were found to be non-differentially expressed genes, indicating that AS serves as another important way to regulate the infection of S. sclerotiorum on plants besides the gene expression level. Taken together, this study provides a genome-wide landscape for the AS of S. sclerotiorum during infection as well as an important resource for further elucidating the pathogenic mechanisms of S. sclerotiorum.

Molecular characterization of NDM-1-producing carbapenem-resistant E. cloacae complex from a tertiary hospital in Chongqing, China.

Background: The aim of this study was to clarify the molecular characterization of NDM-1-producing carbapenem-resistant Enterobacter cloacae complex (CREL) at a teaching hospital in Chongqing, China. Methods: Antimicrobial susceptibility and resistance genes were analyzed. Epidemiological relationship was analyzed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Conjugation experiments were performed to determine the transferability of plasmids. Whole-genome sequencing (WGS) of strains was implemented, and the genetic environment of the bla NDM-1- and mcr-9-carrying plasmids was analyzed. Results: A total of 10 bla NDM-1-positive CREL isolates were identified. All isolates harbored multiple resistance genes. ECL68 and ECL78 co-produce bla NDM-1 and mcr-9. Among the four different sequence types (STs) detected, ST1466 was assigned as a novel ST. Six isolates exhibited highly similar PFGE patterns. Conjugation assay proved that all plasmids containing bla NDM-1 or mcr-9 could be transferred to the recipient Escherichia coli. WGS indicated that bla NDM-1 genes were carried by diverse plasmids, including IncHI2/IncN, IncX3, and one unclassified plasmid type. The backbone structure of these plasmids is involved in replication initiation (repAB), partitioning (parABM), and conjugation/type IV secretion (tra/virB). Analysis of the genetic environment showed that bla NDM-1 in three plasmids exhibited a highly similar structure to protype Tn125. Co-existence of bla NDM-1 and the colistin resistance gene mcr-9 was detected in the two isolates, ECL68 and ECL78. In ECL68, bla NDM-1 and mcr-9 were present on the same plasmid while located in two separate plasmids in ECL78. The genetic environment of mcr-9 was organized as IS26-wbuC-mcr-9-IS903-pcoS-pcoE-rcnA-rcnR, and the two-component system encoding genes qseC and qseB was not found in two plasmids, which could explain mcr-9-harboring strains' colistin susceptibility. Conclusions: We first report a nosocomial outbreak of NDM-1-producing E. cloacae complex ST177 in China. Conjugative plasmids contributed to the horizontal transfer of antibiotic resistance genes. The prevalence and even coexistence of bla NDM-1 and mcr-9 may further threaten public health. Our results highlight further surveillance for bla NDM-1, and mcr-9 is essential to prevent its dissemination.

Increased Expression and Amplification of blaKPC-2 Contributes to Resistance to Ceftazidime/Avibactam in a Sequence Type 11 Carbapenem-Resistant Klebsiella pneumoniae Strain.

Ceftazidime/avibactam (CAZ/AVI) is regarded as an effective alternative antibiotic for the clinical treatment of Klebsiella pneumoniae carbapenemase (KPC)-producing isolates. As resistance has been reported in some strains, it is critical to understand the key mechanisms contributing to the acquired resistance to CAZ/AVI. From January 2018 to April 2020, 127 KPC-producing carbapenem-resistant Klebsiella pneumoniae strains (CRKPs) were isolated at a university hospital in Chongqing, China, and 25 strains showed reduced susceptibility to CAZ/AVI. All reduced-susceptibility CRKPs were deficient in Ompk35 and Ompk36 porins, and 24 strains had a premature termination at amino acid position 63 in Ompk35 and 134 to 135 glycine and aspartic acid (GD) insertion in OmpK36, while the blaKPC-2 expression level showed no significant difference compared to that of strain BAA-1705. Four reduced-susceptibility strains evolved resistance under selective pressure of CAZ/AVI with the blaKPC-2 expression level increased, and two of these strains had mutations in the Ω-loop. The study found a strain of CRKP55 with changes in the resistance phenotype during conjugation, evolving from reduced sensitivity to high-level resistance to CAZ/AVI. Through plasmid sequencing and reverse transcription-quantitative PCR, it was speculated that insertion sequence (IS)26-mediated blaKPC-2 gene amplification caused the MIC value change in the conjugant JKP55. Our findings illustrated the potential of CAZ/AVI resistance under antibiotic stress and demonstrated that IS26 may mediate blaKPC-2 replication transposition, leading to high-level resistance during horizontal gene transfer. Investigation of CAZ/AVI resistance mechanisms may offer a unique opportunity to study the horizontal evolutionary trajectories of K. pneumoniae high-risk clones. IMPORTANCE Klebsiella pneumoniae carbapenemase (KPC) production is the most common mechanism of K. pneumoniae resistance to carbapenems in China. Currently, CAZ/AVI is considered a potential alternative therapeutic option for infections caused by these isolates. However, there have been increasing reports of resistant or reduced-sensitivity strains since the approval of this agent. In this study, resistance to CAZ/AVI was induced under drug-selective pressure and was caused by blaKPC-2 overexpression and/or substitutions in the Ω-loop of KPC. Additionally, it was demonstrated that a conjugative plasmid carrying blaKPC-2 could transfer horizontally between species, and perhaps, IS26-derived tandem amplification of blaKPC-2 during this period led to high-level resistance to CAZ/AVI. Our research suggests that IS26-mediated resistance evolution may have important implications in guiding clinical antibiotic use.

Room-Temperature Solid-State UV Cross-Linkable Vitrimer-like Polymers for Additive Manufacturing.

In this paper, a UV cross-linkable vitrimer-like polymer, ureidopyrimidinone functionalized telechelic polybutadiene, is reported. It is synthesized in two steps. First, 2(6-isocyanatohexylaminocarbonylamino)-6-methyl-4[1H]-pyrimidinone (UPy-NCO) reacts with hydroxy-functionalized polybutadiene (HTPB) to obtain UPy-HTPB-UPy, and then the resulted UPy-HTPB-UPy is cross-linked under 365 nm UV light (photo-initiator: bimethoxy-2-phenylacetophenone, DMPA). Further investigation reveals that the density of cross-linking and mechanical properties of the resulting polymers can be tailored via varying the amount of photo-initiator and UV exposure time. Before UV cross-linking, UPy-HTPB-UPy is found to be vitrimer-like due to the quadruple hydrogen-bonding interactions. The UPy groups at the end of the chain also enable for rapid solidification upon the evaporation of the solvent. The unsaturated double bonds in the HTPB chains enable UPy-HTPB-UPy to be UV cross-linkable in the solid state at room temperature. After cross-linking, the polymers have good shape memory effect (SME). Here, we demonstrate that this type of polymer can have many potential applications in additive manufacturing. In the cases of fused deposition modelling (FDM) and direct ink writing (DIW), not only the strength of the interlayer bonding but also the strength of the polymer itself can be enhanced via UV exposure (from thermoplastic to thermoset) either during printing or after printing. The SME after cross-linking further helps to achieve rapid volumetric additive manufacturing anytime and anywhere.

SnRK1.1-mediated resistance of Arabidopsis thaliana to clubroot disease is inhibited by the novel Plasmodiophora brassicae effector PBZF1.

Plants have evolved a series of strategies to combat pathogen infection. Plant SnRK1 is probably involved in shifting carbon and energy use from growth-associated processes to survival and defence upon pathogen attack, enhancing the resistance to many plant pathogens. The present study demonstrated that SnRK1.1 enhanced the resistance of Arabidopsis thaliana to clubroot disease caused by the plant-pathogenic protozoan Plasmodiophora brassicae. Through a yeast two-hybrid assay, glutathione S-transferase pull-down assay, and bimolecular fluorescence complementation assay, a P. brassicae RxLR effector, PBZF1, was shown to interact with SnRK1.1. Further expression level analysis of SnRK1.1-regulated genes showed that PBZF1 inhibited the biological function of SnRK1.1 as indicated by the disequilibration of the expression level of SnRK1.1-regulated genes in heterogeneous PBZF1-expressing A. thaliana. Moreover, heterogeneous expression of PBZF1 in A. thaliana promoted plant susceptibility to clubroot disease. In addition, PBZF1 was found to be P. brassicae-specific and conserved. This gene was significantly highly expressed in resting spores. Taken together, our results provide new insights into how the plant-pathogenic protist P. brassicae employs an effector to overcome plant resistance, and they offer new insights into the genetic improvement of plant resistance against clubroot disease.

An Outbreak of Carbapenem-Resistant Klebsiella pneumoniae in an Intensive Care Unit of a Major Teaching Hospital in Chongqing, China.

Background: Due to the critical condition and poor immunity of patients, the intensive care unit (ICU) has always been the main hospital source of multidrug-resistant bacteria. In recent years, with the large-scale use of antibiotics, the detection rate and mortality of carbapenem-resistant Klebsiella pneumoniae (CRKP) have gradually increased. This study explores the molecular characteristics and prevalence of CRKP isolated from the ICU ward of a tertiary hospital in China. Methods: A total of 51 non-duplicated CRKP samples isolated from the ICU were collected from July 2018-July 2020. The enzyme production of the strains was preliminarily screened by carbapenemase phenotypic test, and drug-resistant and virulence genes were detected by PCR. The transferability of plasmid was verified by conjugation test. The minimal inhibitory concentration (MIC) was determined by microbroth dilution method and genetic diversity was detected by multilocus sequence typing and pulsed-field gel electrophoresis. Results: blaKPC-2 was the only carbapenemase detected. The major virulence genes were uge (100%), mrkD (94.1%), kpn (94.1%), and fim-H (72.5%), while wcag, ironB, alls and magA genes were not detected. One sequence type ST1373 strain, hypervirulent K. pneumoniae (hvKP), was detected. CRKP strains were highly resistant to quinolones, cephalosporins, aminoglycosides, and polymyxin, but susceptive to tigecycline and ceftazidime-avibactam. The success rate of conjugation was 12.2%, indicating the horizontal transfer of blaKPC-2 . Homology analysis showed that there was a clonal transmission of ST11 CRKP in the ICU of our hospital. Conclusion: The present study showed the outbreak and dissemination in ICU were caused by ST11 CRKP, which were KPC-2 producers, and simultaneously, also carried some virulence genes. ST11 CRKP persisted in the ward for a long time and spread among different areas. Due to the widespread dispersal of the transferable blaKPC-2 plasmid, the hospital should promptly adopt effective surveillance and strict infection control strategies to prevent the further spread of CRKP. Ceftazidime-avibactam showed high effectiveness against CRKP and could be used for the treatment of ICU infections.

Carbapenemase Production and Epidemiological Characteristics of Carbapenem-Resistant Klebsiella pneumoniae in Western Chongqing, China.

Background: This study aimed to determine the molecular characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in a hospital in western Chongqing, southwestern China. Methods: A total of 127 unique CRKP isolates were collected from the Yongchuan Hospital of Chongqing Medical University, identified using a VITEK-2 compact system, and subjected to microbroth dilution to determine the minimal inhibitory concentration. Enterobacteriaceae intergenic repeat consensus polymerase chain reaction and multilocus sequence typing were used to analyze the homology among the isolates. Genetic information, including resistance and virulence genes, was assessed using polymerase chain reaction. The genomic features of the CRKP carrying gene blaKPC-2 were detected using whole-genome sequencing. Results: ST11 was the dominant sequence type in the homology comparison. The resistance rate to ceftazidime-avibactam in children was much higher than that in adults as was the detection rate of the resistance gene blaNDM (p < 0.0001). Virulence genes such as mrkD (97.6%), uge (96.9%), kpn (96.9%), and fim-H (84.3%) had high detection rates. IncF (57.5%) was the major replicon plasmid detected, and sequencing showed that the CRKP063 genome contained two plasmids. The plasmid carrying blaKPC-2, which mediates carbapenem resistance, was located on the 359,625 base pair plasmid IncFII, together with virulence factors, plasmid replication protein (rep B), stabilizing protein (par A), and type IV secretion system (T4SS) proteins that mediate plasmid conjugation transfer. Conclusion: Our study aids in understanding the prevalence of CRKP in this hospital and the significant differences between children and adults, thus providing new ideas for clinical empirical use of antibiotics.

Microbial Characteristics and Genomic Analysis of an ST11 Carbapenem-Resistant Klebsiella pneumoniae Strain Carrying bla KPC-2 Conjugative Drug-Resistant Plasmid.

BACKGROUND: The sequence type 11 (ST11) carbapenem-resistant Klebsiella pneumoniae (CRKP) carrying bla KPC-2 has been widespread all over the world, and it has been reported frequently in China. The bla KPC-2 located on the mobile genetic element brings tremendous pressure to control the spread and outbreak of resistant bacteria. Whole-genome sequencing (WGS) technology can comprehensively and in-depth display the molecular characteristics of drug-resistant bacteria, providing a basis for evaluating the genetic diversity within the CRKP genome. METHODS: The ST11 CRKP in this study was collected in the intensive care unit of a major teaching hospital. PCR and Sanger sequencing confirmed the existence of bla KPC-2. The AST-GN card and the microbroth dilution test were used for antimicrobial susceptibility testing. The transferability of plasmid was verified by a conjugation test. The whole genome is sequenced using the Illumina HiSeq short-read and Oxford Nanopore long-read sequencing technology. RESULTS: The studied strain was named CRKP63, which is a multi-drug resistance bacteria, which carries bla KPC-2 and bla SHV-182. Its genome consists of a circular chromosome of 5,374,207 bp and an IncFII plasmid named pKPC-063001 of 359,625 bp. In the drug-resistant plasmid pKPC-063001, the key carbapenem resistance gene bla KPC-2 was located in the genetic context with insertion sequence ISKpn27 upstream and ISKpn6 downstream and bracketed by IS26. The three copies of the IS26-ISKpn27-bla KPC-2-ISKpn6-IS26 unit were present in tandem. bla KPC-2 can be transferred horizontally between other species by conjugation, the complete type IV secretion system (T4SS) structure helps to improve the adaptability of bacteria to the external environment, strengthen the existence of drug-resistant bacteria, and accelerate the spread of drug resistance. CONCLUSION: High-throughput sequencing has discovered the different surrounding environments of bla KPC-2, which provides a new idea for further revealing the transmission and inheritance of bla KPC-2 at the molecular level. In order to control the further spread and prevalence of drug-resistant bacteria, we should pay close attention to the changes in the genetic environment of bla KPC-2 and further study the transcription and expression of T4SS.

Antibiotic susceptibility and molecular analyses of clinical Enterobacter cloacae isolates in Eastern Heilongjiang Province, China.

BACKGROUND: Enterobacter cloacae is an emerging opportunistic pathogen. We retrospectively conducted a study to assess antimicrobial susceptibility and investigated the Molecular characteristics of carbapenem-resistant Enterobacter cloacae (CREL) isolates. METHODS: Three hundred forty-two isolates of Enterobacter cloacae were collected from January 2014 to December 2018. Ten strains of CREL were collected for further research. The species identifications and minimum inhibitory concentrations (MICs) of all antibiotics tested were analyzed using the Vitek 2 Compact system (BioMerieux, France) and supplemented by the disk diffusion method. Polymerase chain reaction (PCR) was performed to detect extended-spectrum ß-lactamase (ESBL) and carbapenemase resistance genes. RESULTS: The results showed that most of the isolates remained susceptible to tested antibiotics; however, the resistance rate of Cefepime has been increasing in recent years. One strain co-producing New Delhi Metallo-ß-lactamase NDM-1 and Imipenem hydrolase IMP-4. NDM-1 and IMP-4-producing isolates highlight that active surveillance is necessary to prevent the further spread of the bacteria. Multilocus sequence typing (MLST) showed that two KPC-producing isolates assigned to ST93, two isolates carrying NDM-1 assigned to ST1120. Moreover, the MEGA analysis showed that ST93, ST256, and ST1120 have homology, showing that CREL in our area has a potential spread risk. CONCLUSIONS: These findings indicating that CREL clonal dissemination may occurred in this region and should be taken seriously concern. Our study highlights an urgent need to monitor these isolates to prevent their further spread.

The determination of gyrA and parC mutations and the prevalence of plasmid-mediated quinolone resistance genes in carbapenem resistant Klebsiella pneumonia ST11 and ST76 strains isolated from patients in Heilongjiang Province, China.

BACKGROUND: There is increasing resistance to carbapenems among Klebsiella pneumoniae,and fluoroquinolones (FQ) are increasingly used to treat infections from extended-spectrum ß- lactamase(ESBLs) and carbapenemase-producing Klebsiella pneumoniae. However, the acquisition of plasmid-mediated quinolone resistance (PMQR) or the spontaneous mutation of the quinolone resistance-determining regions (QRDR) of the gyrA and parC genes can severely affect the therapeutic effect of quinolones. The goal of this study was to investigate the molecular determinants of FQ resistance(FQ-R) in carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates from Heilongjiang Province,China. MATERIALS AND METHODS: We isolated 40 strains of CRKP from a treatment center in the eastern part of Heilongjiang Province from January 2016 to December 2018. The VITEK2 Compact analyzer was used to identify and detect drug sensitivity. Different types of drug resistance genes were detected by polymerase chain reaction (PCR). PCR and DNA sequencing were used to assess the presence of qnrA, qnrB, qnrS,qepA and acc(6') Ib-cr genes,which are plasmid-encode genes that can contribute to resistance. The sequences of gyrA and parC genes were sequenced and compared with the sequences of standard strains to determine if mutations were present.Multi-site sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed on the strains to assess homology. RESULTS: The isolated CRKP strains showed rates of resistance to fluoroquinolones of 22.5% to 42.5%. The resistance rate of ciprofloxacin was significantly higher than that of levofloxacin.Nine CRKP strains (22.5%) showed co-resistance to ciprofloxacin and levofloxacin.The quinolone resistant strains were screened for plasmid-encoded genes that can contribute to resistance (PMQR genes).Among the 17 quinolone resistant strains,one strain contained no PMQR genes,twelve strains contained two PMQR genes,and four strains contained four PMQR genes.Acc (6') Ib-cr was the most frequently detected PMQR gene, detected in 95% of strains tested (38 of 40) and in 94.1% of the quinolone-resistant strains (16 of 17). The qepA gene encoding an efflux pump was not detected in any strains.No isolate carried five different PMQRs simultaneously.Changes of S83I and D87G changes in gyrA, and the S80I change in parC,which were mediated by QRDR,were identified in two isolates,which showed resistance to both ciprofloxacin and levofloxacin.Most of the FQ-R strains(58.8%,10/17) belong to ST(sequence type) 76, which is dominant in the local area, while all the mutant strains (100%,2/2),that differ in at least one site from standard bacteria, belong to the ST11 group. The strains were isolated from a hospital where there had been a recent outbreak of ST76 type CRKP in the neurosurgery ward and intensive care unit. CONCLUSION: CRKP strains were identified that were insensitive or even resistant to quinolones,and this resistance is common in Heilongjiang Province of eastern China;fluoroquinolone-resistance in these clinical CRKP strains is a complex interplay between PMQR determinants and mutations in gyrA and parC.The resistance level caused by QRDR mutation is higher than that caused by PMQR, however, the high frequency of PMQR genes in the isolated CRKP strains suggests the potential for impact of these genes.PMQR determinants are often found in carbapenemase-producing or ESBLs-producing Klebsiella pneumoniae,and some resistance genes,such as:SHV,TEM, CTX-M-15,and OXA-1 are closely associated with FQ-R. Finally, geographical factors can affect the emergence and spread of PMQR and QRDR.Some genetic lineages have higher potential risks, and continuous close monitoring is required.

Identification and Characterization of Plasmodiophora brassicae Primary Infection Effector Candidates that Suppress or Induce Cell Death in Host and Nonhost Plants.

Clubroot caused by Plasmodiophora brassicaeis one of the most important diseases in cruciferous crops. The recognition of P. brassicae by host plants is thought to occur at the primary infection stage, but the underlying mechanism remains unclear. Secretory proteins as effector candidates play critical roles in the recognition of pathogens and the interactions between pathogens and hosts. In this study, 33 P. brassicae secretory proteins expressed during primary infection were identified through transcriptome, secretory protein prediction, and yeast signal sequence trap analyses. Furthermore, the proteins that could suppress or induce cell death were screened through an Agrobacterium-mediated plant virus transient expression system and a protoplast transient expression system. Two secretory proteins, PBCN_002550 and PBCN_005499, were found to be capable of inducing cell death associated with H2O2 accumulation and electrolyte leakage in Nicotiana benthamiana. Moreover, PBCN_002550 could also induce cell death in Chinese cabbage. In addition, 24 of the remaining 31 tested secretory proteins could suppress mouse Bcl-2-associated X protein-induced cell death, and 28 proteins could suppress PBCN_002550-induced cell death.

Jasmonic Acid-Mediated Aliphatic Glucosinolate Metabolism Is Involved in Clubroot Disease Development in Brassica napus L.

Glucosinolate (GSL) is associated with clubroot disease, which is caused by the obligate biotrophic protist Plasmodiophora brassicae. Due to the complicated composition of GSLs, their exact role in clubroot disease development remains unclear. By investigating clubroot disease resistance in cruciferous plants and characterizing the GSL content in seeds, we can determine if clubroot disease development is related to the components of GSLs. The difference in the infection process between Matthiola incana L. (resistant) and Brassica napus L. (susceptible) was determined. Root hair infection was definitely observed in both resistant and susceptible hosts, but no infection was observed during the cortical infection stage in resistant roots; this finding was verified by molecular detection of P. brassicae via PCR amplification at various times after inoculation. Based on the time course detection of the contents and compositions of GSLs after P. brassicae inoculation, susceptible roots exhibited increased accumulation of aliphatic, indolic, and aromatic GSLs in B. napus, but only aromatic GSLs were significantly increased in M. incana. Gluconapin, which was the main aliphatic GSL in B. napus and present only in B. napus, was significantly increased during the secondary infection stage. Quantification of the internal jasmonic acid (JA) concentration showed that both resistant and susceptible plants exhibited an enhanced level of JA, particularly in susceptible roots. The exogenous JA treatment induced aliphatic GSLs in B. napus and aromatic GSLs in M. incana. JA-induced aromatic GSLs may be involved in the defense against P. brassicae, whereas aliphatic GSLs induced by JA in B. napus likely play a role during the secondary infection stage. Three candidate MYB28 genes regulate the content of aliphatic GSLs identified in B. napus; one such gene was BnMYB28.1, which was significantly increased following both the treatment with exogenous JA and P. brassicae inoculation. In summary, the increased content of JA during the secondary infection stage may induce the expression of BnMYB28.1, which caused the accumulation of aliphatic GSLs in clubroot disease development.